LOCUS       sequence-116406-        5912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked SpHIS5 marker, integration into S. cerevisiae XII-4 
            chromosomal location, USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5912)
  AUTHORS   Stovicek V, Borja GM, Forster J, Borodina I
  TITLE     EasyClone 2.0: expanded toolkit of integrative vectors for stable 
            gene expression in industrial Saccharomyces cerevisiae strains.
  JOURNAL   J Ind Microbiol Biotechnol. 2015 Sep 16.
  PUBMED    26376869
REFERENCE   2  (bases 1 to 5912)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Ind 
            Microbiol Biotechnol. 2015 Sep 16."
COMMENT     Sequence Label: pCfB2228
FEATURES             Location/Qualifiers
     source          1..5912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      30..219
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    267..300
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            355..1555
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        355..698
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             699..1352
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe his5"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=S. pombe his5
                     /note="yeast auxotrophic marker; corresponds to S. 
                     cerevisiae HIS3"
                     /translation="MGRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHANQ
                     KGEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQ
                     AMGNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLY
                     SFSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     terminator      1358..1555
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1615..1648
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1691..1710)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1692..1710)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1720..1736)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1729..1751)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2356..2389)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(2859..2875)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2974..2991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3145..3733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3225..3244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3857..4648,4649..4717))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4480..4499
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4718..4809)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4851..4870
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4876..4895
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5102..5135
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     terminator      complement(5718..5883)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5804..5828
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
ORIGIN
        1 caacggaatg cgtgcgatcg cgtgcattca tccgctctaa ccgaaaagga aggagttaga
       61 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta
      121 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata
      181 ctgaaaacct tgcttgagaa ggttttggga cgctcgaaga tcgcgtcagc tgaagcttcg
      241 tacgctgcag gtcgacaacc cttaatataa cttcgtataa tgtatgctat acgaagttat
      301 taggtctaga gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg
      361 gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac
      421 tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt
      481 gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga
      541 aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata
      601 aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta
      661 ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaggagg gcttttgtag
      721 aaagaaatac gaacgaaacg aaaatcagcg ttgccatcgc tttggacaaa gctcccttac
      781 ctgaagagtc gaattttatt gatgaactta taacttccaa gcatgcaaac caaaagggag
      841 aacaagtaat ccaagtagac acgggaattg gattcttgga tcacatgtat catgcactgg
      901 ctaaacatgc aggctggagc ttacgacttt actcaagagg tgatttaatc atcgatgatc
      961 atcacactgc agaagatact gctattgcac ttggtattgc attcaagcag gctatgggta
     1021 actttgccgg cgttaaaaga tttggacatg cttattgtcc acttgacgaa gctctttcta
     1081 gaagcgtagt tgacttgtcg ggacggccct atgctgttat cgatttggga ttaaagcgtg
     1141 aaaaggttgg ggaattgtcc tgtgaaatga tccctcactt actatattcc ttttcggtag
     1201 cagctggaat tactttgcat gttacctgct tatatggtag taatgaccat catcgtgctg
     1261 aaagcgcttt taaatctctg gctgttgcca tgcgcgcggc tactagtctt actggaagtt
     1321 ctgaagtccc aagcacgaag ggagtgttgt aaagagtact gacaataaaa agattcttgt
     1381 tttcaagaac ttgtcatttg tatagttttt ttatattgta gttgttctat tttaatcaaa
     1441 tgttagcgtg atttatattt tttttcgcct cgacatcatc tgcccagatg cgaagttaag
     1501 tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc
     1561 gattcgatac taacgccgcc atccagttta aacgagctct cgagaaccct taatataact
     1621 tcgtataatg tatgctatac gaagttatta ggtgatatca gatccactag tggcctatgc
     1681 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     1741 tcgtgactgg gaaaaccctg gcgttacccc tgcaggacta gtgctgaggc attaatccaa
     1801 ctttgtacta ttccttcccc gtttactcaa ttcttgaagc caatttgtac aattccccat
     1861 tagagtcaaa taaaaggatg cctcacggag gtatgttacc cgcgctattt cacatggctc
     1921 attgaattag aggtggaatt tggtgtaccc tcccctcctc atctgatgaa gtagtgatcc
     1981 gacaattctt aaaagttgta gacattactt ttaccaccaa ctaagttgta tttatattgc
     2041 tacccttatc cttttatatc taactagcgc tcataaggtt ggggcaatac taaaactgtg
     2101 ttcttattca actcattaaa tacgtggcag tacgtaccct attagaaaca ataggaaaca
     2161 gcagagtcgg aagaagccaa atgccagatt tgaagtccaa aaccttgtca agccaatctt
     2221 tgggagcggc tattcctcca gaaattgtgt accaaatact tacataccag tttagggatt
     2281 tgttaagaaa tgaccatcca ggtacggcag aaaattaaga cctcagcgcg gccgcaaatt
     2341 taaataaaat gaagtgaagt tcctatactt tctagagaat aggaacttct atagtgagtc
     2401 gaataagggc gacacaaaat ttattctaaa tgcataataa atactgataa catcttatag
     2461 tttgtattat attttgtatt atcgttgaca tgtataattt tgatatcaaa aactgatttt
     2521 ccctttatta ttttcgagat ttattttctt aattctcttt aacaaactag aaatattgta
     2581 tatacaaaaa atcataaata atagatgaat agtttaatta taggtgttca tcaatcgaaa
     2641 aagcaacgta tcttatttaa agtgcgttgc ttttttctca tttataaggt taaataattc
     2701 tcatatatca agcaaagtga caggcgccct taaatattct gacaaatgct ctttccctaa
     2761 actcccccca taaaaaaacc cgccgaagcg ggtttttacg ttatttgcgg attaacgatt
     2821 actcgttatc agaaccgccc agggggcccg agcttaagac tggccgtcgt tttacaacac
     2881 agaaagagtt tgtagaaacg caaaaaggcc atccgtcagg ggccttctgc ttagtttgat
     2941 gcctggcagt tccctactct cgccttccgc ttcctcgctc actgactcgc tgcgctcggt
     3001 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3061 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3121 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3181 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3241 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3301 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3361 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3421 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3481 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3541 tacagagttc ttgaagtggt gggctaacta cggctacact agaagaacag tatttggtat
     3601 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3661 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3721 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3781 cgcgcgcgta actcacgtta agggattttg gtcatgagct tgcgccgtcc cgtcaagtca
     3841 gcgtaatgct ctgcttttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3901 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3961 gcttaccatc tggccccagc gctgcgatga taccgcgaga accacgctca ccggctccgg
     4021 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4081 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4141 ttaatagttt gcgcaacgtt gttgccatcg ctacaggcat cgtggtgtca cgctcgtcgt
     4201 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4261 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4321 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4381 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4441 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4501 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4561 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4621 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4681 agggaataag ggcgacacgg aaatgttgaa tactcatatt cttccttttt caatattatt
     4741 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4801 ataaacaaat aggggtcagt gttacaacca attaaccaat tctgaacatt atcgcgagcc
     4861 catttatacc tgaatatggc tcataacacc ccttgtttgc ctggcggcag tagcgcggtg
     4921 gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg
     4981 gggactcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
     5041 gaaagactgg gcctttcgcc cgggctaatt atggggtgtc gcccttattc gactctatag
     5101 tgaagttcct attctctaga aagtatagga acttctgaag tggggattta aatgcggccg
     5161 cgctgagggt ttaatgtatc cggctgttcc ttcatagccc tttcaatgaa cgttgcagcc
     5221 ctttgaagat tggccatttt gtcaggactc gagcctgaca gttggaccaa cgcaacttta
     5281 attttttgtg aaagaatctt cgaagcactc atactggcga tcttcacgcc ctcctgctat
     5341 tacaaaagct gtgtttttac aagaatcaaa ttaagttagc aagatattat acaacattat
     5401 tgataatttc aatatcgtgt tcgtacctga tgacgtatct gtgcattgat aaggcccgca
     5461 tggtttcaga aagcagagcg gaacgattcc aaattagtgg ccttgtgctt tgcatgtcaa
     5521 ttgtgttacc ttcagctcgt ggatttgttt tatcaataca cagtctacag tcaagaattt
     5581 ttttttatca aattttgcgt tcgagcgtat aaaatagccg ctgtagctac ttaagttcct
     5641 gttcagcgat agtttttttc catcacacgt actatggcaa ttaagtcctc agcgagctcg
     5701 catggaatgc gtgcgatgag cgacctcatg ctatacctga gaaagcaacc tgacctacag
     5761 gaaagagtta ctcaagaata agaattttcg ttttaaaacc taagagtcac tttaaaattt
     5821 gtatacactt atttttttta taacttattt aataataaaa atcataaatc ataagaaatt
     5881 cgcttattta gaagtgtcaa caacgtatct ac
//
